Ultrasonic-Assisted Extraction of Xanthorrhizol from Curcuma xanthorrhiza Roxb. Rhizomes by Natural Deep Eutectic Solvents: Optimization, Antioxidant Activity, and Toxicity Profiles.

Xanthorrhizol, an important marker of Curcuma xanthorrhiza, has been recognized for its different pharmacological activities. A green strategy for selective xanthorrhizol extraction is required. Herein, natural deep eutectic solvents (NADESs) based on glucose and organic acids (lactic acid, malic acid, and citric acid) were screened for the extraction of xanthorrhizol from Curcuma xanthorrhiza. Ultrasound-assisted extraction using glucose/lactic acid (1:3) (GluLA) gave the best yield of xanthorrhizol. The response surface methodology with a Box-Behnken Design was used to optimize the interacting variables of water content, solid-to-liquid (S/L) ratio, and extraction to optimize the extraction. The optimum conditions of 30% water content in GluLA, 1/15 g/mL (S/L), and a 20 min extraction time yielded selective xanthorrhizol extraction (17.62 mg/g) over curcuminoids (6.64 mg/g). This study indicates the protective effect of GluLA and GluLA extracts against oxidation-induced DNA damage, which was comparable with those obtained for ethanol extract. In addition, the stability of the xanthorrhizol extract over 90 days was revealed when stored at -20 and 4 °C. The FTIR and NMR spectra confirmed the hydrogen bond formation in GluLA. Our study reported, for the first time, the feasibility of using glucose/lactic acid (1:3, 30% water v/v) for the sustainable extraction of xanthorrhizol.

Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system.

Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.

Gold nanoparticles (AuNP)-based aptasensor for enteropathogenic Escherichia coli detection.

BACKGROUND: Diarrhea is a major cause of severe gastrointestinal illness in the infant especially in many developing countries. Although this molecular technique has been accepted as standard technique to detect Diarrhea-causing EPEC, the practical aspect of this technique for in-site rapid screening purposes is still facing a major challenge. In this study, we characterized EPEC specific aptamers and applied it as an AuNP-based aptasensor for point of care (POC) diagnosis purpose. METHODS: As many as six selected DNA aptamers was screened using target bacteria and the bound aptamer was measured by qPCR technique. Moreover, Kd value for each optimal bound aptamer was measured by using the same technique. Colorimetry assay was applied to test specificity and LOD of AuNP-based aptasensor. RESULTS: Two DNA aptamers have been successfully obtained to detect Enteropathogenic Escherichia coli K.1.1. DNA aptamer S8-7 exhibited constant dissociation (Kd) value of 17.08 nM, while DNA aptamer S10-5 exhibited Kd value of 34.14 nM. AuNP-based aptasensor showed high selectivity and specificity for EPEC K.1.1 with a limit of detection (LOD) value of 105 CFU/mL. Truncation study on DNA aptamer S8-7 showed that elimination of primer binding sequence only slightly increased both performance of detection and LOD value of AuNP-based aptasensor. CONCLUSION: Further study is necessary to improve AuNP-aptasensor performance such as through mutagenesis approach on targeted DNA aptamers before AuNP-based aptasensor can be applied as a biosensor in point of care (POC) diagnosis.

Efficacy, toxicity study and antioxidant properties of plantaricin E and F recombinants against enteropathogenic Escherichia coli K1.1 (EPEC K1.1).

Enteropathogenic Escherichia coli (EPEC) is one of the resistance bacteria towards antibiotics and have been raising problem during treatments. Therefore, a new antibiotic candidate is required. Plantaricin E and F recombinant have been successfully produced by a GRAS host Lactococcus lactis. This study was aimed to evaluate the efficacy and toxicity of plantaricin E and F recombinant against EPEC K1.1 infection by in vivo assay. The production of plantaricin E and F recombinants from Lactococcus lactis was conducted and encapsulated. The in vivo study was carried out by inoculating the mice perorally with EPEC K1.1 for 7 days then treated with 100, 250, and 500 mg/kg body weight/day of recombinant plantaricin E and F for another 7 days. The toxicity assay were observed in ddY mice using various concentrations of treatment (50, 100, 1000, and 5000 mg/kg/body weight) doses perorally for 48 h. The result showed that the plantaricin E and F recombinant were successfully produced in Lactococcus lactis expression host with 3.7 kDa and 3.8 kDa in size. The efficacy study revealed the optimal doses of plantaricin E and F recombinant against EPEC K1.1 infection was 250 mg/kgBW for plantaricin E and 500 mg/kgBW for plantaricin F. The plantarisin E and F recombinant treatment showed improvement in leukocyte, hematocrit, and hemoglobin levels as well in decreasing malondialdehyde (MDA) level. Observation of the intestine histopathology showed small amounts of mononuclear inflammatory cell infiltration than the other groups of treatment. The acute toxicity assay showed that there was no mortality observed during the assay, even after 5000 mg/kg body weight of plantarisin E and F recombinant treatment (LD50 > 5000 mg/KgBW). The hematological and biochemical observations showed normal levels in leukocytes, erythrocytes, hematocrit, hemoglobin, platelets, urea, creatinine, and alanine transaminase aspartate transaminase (SGOT and SGPT) while histopathological observation shows a picture of normal liver and kidney cells. This study confirmed the application of bacteriocin for further academic and industrial purposes as a non-toxic substance for food preservative and antibiotic candidate.

Curcumin-derived carbon-dots as a potential COVID-19 antiviral drug.

Even entering the third year of the COVID-19 pandemic, only a small number of COVID-19 antiviral drugs are approved. Curcumin has previously shown antiviral activity against SARS-CoV-2 nucleocapsid, but its poor bioavailability limits its clinical uses. Utilizing nanotechnology structures, curcumin-derived carbon-dots (cur-CDs) were synthesized to increase low bioavailability of curcumin. In-silico analyses were performed using molecular docking, inhibition of SARS-CoV-2 nucleocapsid C-terminal domain (N-CTD) and antiviral activity were assessed in dimer-based screening system (DBSS) and in vitro respectively. Curcumin bound with the N-CTD at ΔG = -7.6 kcal/mol, however modifications into cur-CDs significantly improved the binding affinity and %interaction. Cur-CDs also significantly increased protection against SARS-CoV-2 in both DBSS and in vitro at MOI = 0.1. This study demonstrated the effect of post-infection treatment of curcumin and novel curcumin-derived carbon-dots on SARS-CoV-2 N-CTD dimerization. Further investigation on pre-infection and in-vivo treatment of curcumin and cur-CDs are required for a comprehensive understanding on the carbon-dots enhanced antiviral activity of curcumin against SARS-CoV-2.

Porphyrin-derived carbon dots for an enhanced antiviral activity targeting the CTD of SARS-CoV-2 nucleocapsid.

BACKGROUND: Since effective antiviral drugs for COVID-19 are still limited in number, the exploration of compounds that have antiviral activity against SARS-CoV-2 is in high demand. Porphyrin is potentially developed as a COVID-19 antiviral drug. However, its low solubility in water restricts its clinical application. Reconstruction of porphyrin into carbon dots is expected to possess better solubility and bioavailability as well as lower biotoxicity. METHODS AND RESULTS: In this study, we investigated the antiviral activity of porphyrin and porphyrin-derived carbon dots against SARS-CoV-2. Through the in silico analysis and assessment using a novel drug screening platform, namely dimer-based screening system, we demonstrated the capability of the antivirus candidates in inhibiting the dimerization of the C-terminal domain of SARS-CoV-2 Nucleocapsid. It was shown that porphyrin-derived carbon dots possessed lower cytotoxicity on Vero E6 cells than porphyrin. Furthermore, we also assessed their antiviral activity on the SARS-CoV-2-infected Vero E6 cells. The transformation of porphyrin into carbon dots substantially augmented its performance in disrupting SARS-CoV-2 propagation in vitro. CONCLUSIONS: Therefore, this study comprehensively demonstrated the potential of porphyrin-derived carbon dots to be developed further as a promisingly safe and effective COVID-19 antiviral drug.

Possible intraindividual evolution of SARS-CoV-2 in nasopharyngeal and anal swab in an octogenarian: a case report.

Introduction: COVID-19 is an emerging infectious disease that remains to be further investigated. Case report: Here, we describe a case of COVID-19 in an octogenarian woman with comorbidities who slowly recovered during hospitalization, but died due to sudden cardiac death after 2 weeks of hospitalization. Her nasopharyngeal and anal swabs returned positive for SARS-CoV-2 by RT-PCR on day 7 of hospitalization. The NGS showed possible intraindividual evolution of virus. The sample from the nasopharyngeal swab yielded a B.1470 variant classified as clade GH. This variant showed mutation in the spike gene D614G; N gene; NS3 gene; NSP2 gene and NSP12 gene. The sample from the anal swab showed similar mutation but with additional point mutation in spike gene S12F and was classified as B.1.465 variant. Conclusions: The possibility of the gastrointestinal tract that served as reservoir for virus mutation accumulation should also be considered and the potential impact of viral fecal transmission in the environment should be further investigated.

Removing a Cystein Group On Interferon Alpha 2b at Position 2 and 99 does Not Diminish Antitumor Activity of the Protein, Even Better.

Interferon alpha 2b is the only standard therapeutic protein for hepatitis virus infections. Further study demonstrated that this protein also posseses antitumor activity in several cancerous organs. One main pathway of this antitumor activity is mediated through antiproliferation as well as proapoptotic effects. Previously, we have successfully developed recombinant human interferon alpha 2b (rhIFNα2b) by using a synthetic gene. In addition, two mutein forms of rhIFNα2b were generated to improve the characteristics of this protein. Two point mutations showed better pharmacokinetic profiles than one point mutation as well as the native form. In the present study, this mutein form was studied for ist antitumor effect in vitro using HepG2 cells. As a comparison, the native form as well as a commercial rIFNα2b were used. Several parameters were investigated including the MTT assay, cell viability test, cell cycle using flow cytometric analysis, and the genes and protein expressions involved in cell growth. The latest was observed to study the mechanism of rhIFNα2b. There was no significant difference in the MTT assay and cell viability after cells were treated with both forms of rhIFNα2b. However, the mutein rhIFNα2b tended to show better proapoptotic activity reflected by flow cytometric data, protein expression of pSTAT1, and DNA expression of caspase 3.